Previous work by our group has established deazatetrahydrofolates as a new class of folate anti-metabolites. DDATHF the lead member of the series was designed to be an inhibitor of folate metabolism at a site other than dihydrofolate reductase. DDATHF and several other members of the deazatetrahydrofolate series possess potent in vivo anti-tumor activity. DDATHF itself is about to begin clinical trials. Our previous work has shown that DDATHF exerts its antiproliferative effects through inhibition of the de novo purine synthesis pathway. The present application proposes basic biochemical studies to further elucidate the biologic effects of this exciting new class of agents. There are three specific aims: 1. The mechanism of dezafolate action will be further investigated through inhibition kinetic studies of target enzymes GAR and AICAR transformylase as well as other folate-related enzymes as isolated enzymes in vitro. Effects of deazatetrahydrofolates on purine nucleotide pools, natural folate cofactor pools, and macromolecular synthesis will also be investigated. 2. Factors which determine sensitivity or resistance to deazatetrahydrofolates including transport, polyglutamylation, target enzyme levels, and purine economy will be investigated using DDATHF resistant lines we have developed. We will attempt to establish additional resistant lines with alternate modes of resistance. 3. Structure-activity relationships in the series will be examined by comparing effects of DDATHF diastereomers, 5-deazatetrahydrofolate, 10-formyl-5-deazatetrahydrofolate, acyclo-DDATHF, and 10-hydroxymethyl DDATHF. These studies are essential for the design of future anti-purine drugs and for the optimum clinical use of the entire group of deazatetrahydrofolates.
